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J. Biol. Chem., Vol. 280, Issue 48, 40319-40327, December 2, 2005

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Analysis of Coumarin 7-Hydroxylation Activity of Cytochrome P450 2A6 using Random Mutagenesis^*

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k_cat and K_m) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k_cat/K_m) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher K_m values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations.

Received for publication, July 26, 2005 , and in revised form, September 21, 2005.

^* This work was supported by Grants R01 CA90426, P30 ES00267 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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